We had previously characterized a versatile transporter, DNP-SG ATPase in human tissues, which mediated the AlP-dependent transport of anionic glutathione-conjugates as well as doxorubicin (DOX). Studies in the currently funded project were aimed at structural and functional characterization of DNP-SG ATPase and immunologically related transport proteins with the overall objective of delineating their relevance to MDR. We are pleased to report successful cloning of DNP-SG AlPase, and its functional reconstitution in artificial proteoliposomes. Our studies show that DNP-SG ATPase is identical to RLIP76, a ral-binding GTPase activating effector protein with previously unknown function. Our studies demonstrated that DNP-SG ATPase\RLIP76 is a 76 kDa precursor protein which gives rise to 5 major peptides which can reconstitute an AlP-dependent primary active DOX and glutathione conjugate transporter in artificial liposomes. RLIP76 over-expression confers a drug-accumulation defect and multidrug resistance when expressed in mammalian cells. Antibodies against RLIP76 inhibit DOX transport in lung cancer cells and markedly potentiate its cytotoxicity in a linear, concentration dependent manner at low concentrations. Comparison between SCLC and NSCLC shows that RLI76 is structurally and functionally distinct between SCLC and NSCLC: whereas SCLC express the wild-type RLIP76, NSCLC cells express a variant which has distinct proteolytic susceptibility, greater stability and significantly greater transport activity than RLIP76 from SCLC. Uptake of wild-type RLIP76-proteoliposomes by NSCLC confers a non-adherent morphology similar to that of SCLC. These observations lead us to hypothesize that RLIP76 is a multi-drug resistance-mediating precursor protein for an AlP-dependent transporter composed from its peptide fragments, that RLIP76 in NSCLC is more active than that present in SCLC, and that distinct nature/processing of its peptide fragments contributes to the observed differences in drug resistance and transport between SCLC and NSCLC. We propose to test our hypothesis through specific aims of 1) establishing that RLIP76 is a versatile AlP-dependent transporter of structurally diverse chemotherapeutic drugs, 2) demonstrating structure/function relationships for RLIP76 and its derived peptides, 3) comparing the function of RLIP76 as a drug and GS-E transporter in SCLC and NSCLC, 4) demonstrating that RLIP76 confers an MDR phenotype in lung cancer cells, and 5) demonstrating that limited tryptic peptides of RLIP76 can distinguish SCLC from NSCLC. These observations provide a potentially clinically relevant therapeutic target for lung cancer, and provide fundamental insight into the role in SCLC and NSCLC of drug-transport, signaling and GSH-linked biochemical pathways.